1. Field of the Invention
The field of the invention relates generally to the expression of recombinant DNA. More particularly, the invention relates to novel vectors (and uses therefor) that can be used to express at least three exogenous genes under the control of a single promoter.
2. Background
A persistent problem associated with expression of multiple, individual recombinant polypeptides (i.e. polypeptides that are not fused to each other) via a vector in an expression system is obtaining satisfactory yields of each polypeptide. This is especially true, for example, when the goal is to express multiple proteins that associate with each other upon expression, where poor yield of one or more of the components will hamper or prevent association of the expressed proteins.
The cloning, transformation and expression efficiencies of a vector typically are inversely related to its size, and therefore one common strategy for expressing multiple polypeptides in an expression system is to use multiple vectors instead of “overloading” a single vector. This approach has drawbacks, however. For instance, short of employing a selection protocol for each vector, there is no way to determine with certainty that a cell contains each vector. In addition, vector incompatibility can hinder obtaining suitable expression levels even where there is satisfactory vector uptake by the cells.
A separate approach is to integrate each exogenous gene into a single construct, but under the control of multiple promoters within that construct. This strategy, too, is riddled with disadvantages. For example, obtaining suitable expression requires successful function of multiple promoters, which can be difficult to achieve. Accordingly, there is no way to determine with certainty that a cell contains sufficient levels of each recombinant polypeptide, short of employing a selection protocol for each gene expression product operatively linked to its respective promoter. Furthermore, utilizing one promoter per exogenous gene disadvantageously results in a relatively large vector. Placing all cistrons into a single vector under the control of a single promoter has not been a viable option in nearly all applications, since, e.g., the further a cistron is positioned from its promoter, the less likely is the chance that acceptable expression yields will be obtained for that cistron.
Certain tricistronic vectors are known in the art, however. For example, Burger et al., Appl. Microbiol. Biotechnol. (1999) 52: 345-353 reported a tricistronic vector that encoded, in a 5-prime to 3-prime orientation, (i) a murine light chain Ig, (ii) a murine heavy chain Ig-TNFα fusion and (iii) puromycin acetyltransferase (pac) as a selective marker; Burger et al. stated that the foregoing tricistronic vector was selected because “expression of the selective marker and product are strictly linked” (id. at 351rt. col.).
However, in Burger et al., the non-Ig polypeptide (i.e., pac) functioned only as a selection vehicle and, hence, did not otherwise associate or otherwise interact with either the murine light chain Ig or heavy chain. Ig-TNFα fusion. Accordingly, Burger et al. provides no suggestion that three “structural” polypeptide domains could be expressed in sufficient yields so as to associate or otherwise interact with each other after expression. In other words, the disclosure by Burger et al. did not overcome the prejudice in the art against using a tricistronic vector to express three or more polypeptide domains that associate or otherwise interact with each other subsequent to expression. It is apparent, therefore, that a vector that satisfies these and other drawbacks known in the art is greatly to be desired. The present invention provides such vectors, together with methods for their use.